Under physiological conditions, the ubiquitous isoform of the Na+/H+ exchanger (NHE- 1) mediates the 1:1 exchange of extracellular Na+ for intracellular Ht NHE- 1 is essential for the regulation of cellular volume, growth and intracellular pH. Growth factors and their receptors, G proteins, changes in cell volume, integrins and protein kinases can regulate the activity of NHE- 1, usually by altering the phosphorylation state of NHE- 1 or regulatory proteins, or by inducing protein-protein signaling interactions. Although the activity of NHE- 1 can be regulated by protein phosphorylation and by interactions with other cellular proteins, the specific pathways that lead to these changes have only been partially elucidated. In this continuation application, we propose three specific aims that will explore the mechanisms through which G protein-coupled receptors (GPCR's) rapidly stimulate NHE-l. These aims are extensions of the published and preliminary data that were generated in the last funding period of this grant (7/1/96-1 1/1/00). During that work, we discovered a novel pathway that regulates NHE- 1 activation. This pathway involves the sequential activation of GPCR's of the G, and Gq classes, the tyrosine kinase Jak2, tyrosine phosphorylation of Ca2+/calmodulin (CAM), and binding of CAM to NHE- 1. The aims in which we follow up those findings are as follow: 1. To elucidate the molecular mechanisms through which Jak2 mediates the activation of NHE by GPCR's. 2. To identify and characterize components of the signal transduction complex that regulates NHE activity. 3. To establish the specificity of the regulation of NHE-1 by Jak2 amongst various types of Gj and Gq proteins. To establish the specificity of the Jak2ICAM pathway by performing similar studies in cells that express only single isotypes of NHE. In order to accomplish those aims, we propose to use contemporary methods of cell transfection, biochemistry, biophysics, microscopy and molecular biology to provide novel mechanistic information that will address gaps in our understanding of how NHE- 1 is regulated. We believe that we are uniquely poised to study these novel mechanisms of regulation of NHE- 1, and are enthusiastic about the prospects of continuing these studies.